Treatment options for filling bone voids and promoting bone regeneration at implant sites are currently limited. Native bone consists primarily of collagen I and hydroxyapatite (HA); therefore, a biomaterial consisting of both of these components would closely mimic native bone. Our laboratory is using electrospinning technology to fabricate bone-mimicking scaffolds composed of a synthetic polymer, polycaprolactone (PCL), in combination with collagen I (col) and nanoparticulate hydroxyapatite (HA). Preliminary studies suggest that PCL/col/HA scaffolds are excellent substrates for bone regeneration because they stimulate greater mesenchymal stem cell (MSC) adhesion, spreading, proliferation, and osteoblastic differentiation than scaffolds composed of either PCL or collagen I alone. However, our broad objective is to further enhance the osteoinductivity of these scaffolds by incorporating additional biological modifiers, which in turn will require the development of effective methods for coupling bioactive factors to the scaffold surface. In this proposal we will utilize an HA-binding domain, heptaglutamate (E7), to anchor the osteoinductive factor, Bone Morphogenic Protein-2 (BMP-2), to PCL/col/HA scaffolds, with the goal of stimulating osteoblastic differentiation of MSCs and corresponding, greater in vivo bone formation. As a second objective, we will test the hypothesis that the collagen-derived peptide, DGEA, modified with an E7 domain, can be used as a substitute for native collagen I in inducing osteogenic cell responses, thus eliminating the risks and costs associated with the use of native collagen I. The proposed research will not only facilitate the development of effective bone substitutes but will also have broad impact, given that the E7 domain is expected to increase anchoring of a wide variety of bioactive peptides/proteins to any type of composite biomaterial incorporating HA. Specific Aim 1: E7 domain-directed anchoring of BMP-2 protein/peptides, or BMP-2 -expressing adeno- associated viral vectors, on tri-component scaffolds. Recombinant BMP-2, or a bioactive peptide derived from BMP-2, will be engineered to contain an E7 domain and then coupled to PCL/col/HA scaffolds and evaluated for in vitro osteoblastic differentiation and in vivo bone formation. In addition, we will determine whether the E7 domain can be utilized to deliver BMP-2-expressing gene therapy viral vectors on scaffolds. Specific Aim 2: Use of E7-modified DGEA peptides as a substitute for collagen I in electrospun scaffolds. We hypothesize that PCL/HA scaffolds functionalized with the E7-modified DGEA collagen-mimetic peptide will have similar effects on MSC behavior as the tri-component PCL/col/HA scaffolds, thus providing an alternative to the use of native collagen I. In this aim, E7-DGEA-modified PCL/HA scaffolds will be directly compared with PCL/col/HA scaffolds for the capacity to induce MSC attachment, spreading, proliferation, and in vitro osteoblastic differentiation, as well as in vivo bone formation following implantation into rat craniae. PUBLIC HEALTH RELEVANCE: Work to improve options for healing and regenerating bone continues due to the lack of an optimal treatment regime. This proposal addresses this concern by focusing on a method that could be adapted to new technologies and those already in place, since hydroxyapatite (in one form or another) is widely used not only in bone grafting procedures but also as a coating for metal implants. This research, focusing on the E7 domain as a delivery tool specific to HA, could be used to facilitate longer and more reliable tethering of any number of bioactive peptides and proteins to HA containing biomaterials.